Despite having the ability to detect low copy number XMRV DNA in a large background of cellular DNA, none of the PCR assays found any evidence of XMRV infection in blood cells from patients or controls. Further, no anti-XMRV antibodies were detected, ruling out possible low level or abortive infections in blood or in other reservoirs.

Marco
Personally though, while its a legitimate concern, I'm getting a little hacked off with this constant raising of contamination. Are we going to spend the next 10 years totting up the number of positive v negative studies and raising the spectre of contamination every time XMRV is discussed in a seminar situation?

....In a way, I just don't get it. One group of researchers (WPI, Singh, Kate Bishop who surprisingly also co-authored one of the negative studies) seem to be pressing ahead on the assumption that the positive findings are beyond reproach and moving on to investigate pathology, transmission etc. Others seem stuck at the for/against/contamination stage.

They should all have access to the same published studies including, for example, the correspondence between WPI and Weiss/McClure. Do the likes of Grice et al actually read this stuff before shouting contamination or is contamination one of those words virologists like to drop into conversation to prove how open and objective they are. Could they be suffering from a case of professional jealousy or NIH syndrome? On the other hand, have they read the papers and come to different conclusions?

Marco, I completely agree. It is hard to distinguish professional, objective cautiousness from purposeful, ill-intentioned obfuscation. However, there are subtle differences. Primary among them is taking real action to answer the question scientifically. At its core, the question of contamination is a scientific hypothesis that can be tested. Yet so far all of the proponents of contamination seem to be doing a lot of talking and not much real investigation (aside from a few tests devised by Switzer for the PNAS paper that all failed to detect contamination). If people like McClure were serious scientists concerned about contamination, then they would be proactively conducting collaborative research to get to the bottom of the issue rather than just continually talking about it.

With each passing day and each new half-baked, unsubstantiated claim of contamination it's looking more and more like a case of "If we say it loud enough and often enough it will become true." The sad thing, as you allude to, is that this method is painfully powerful when it comes to stifling real research and progress.

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Yes I agree with both of you. It is starting to irritate me as well as it seems more about mud-slinging than making reasoned arguments about the science of it. I wish it would get sorted for once and for all.

i) until not that long ago, experiments were conducted with looser controls than now, especially during the Cold War era. They were generally, I believe not intended to cause harm, but they also did not go through layers of independent review.

ii) some dangerous experiments have been publicly admitted, with unfortunate consequences for those involved. LSD experiments on unknowing subjects, electroshock, and the famous syphilis trials. This shows that in some cases it was experiments were done on people without their consent or knowledge that they had been experimented on. While there were not many of these, it would be naive to believe that they do not exist until declassified and publicized

iii) and finally a factor I believe is underestimated: a combination of arrogance, ignorance and "whoops who'da thunk that?". Unintended consequences. Examples of possibilities which I believe are reasonable, not nut-case theories:

A bio-agent test is intended to cause a few sneezes and test only if the powder disperses to the population, but some percent of the public becomes violently sick, someone gets dizzy and falls off a subway platform. None of the healthy subjects they tested the "harmless" substance on had such a reaction -- they didn't bother considering the full range of people and effects involved.

Or as you suggest, a substance beleived to be mostly benign is prepared at a remote test location where bio-weopons are developed. It isn't thoroughly tested and contains a contaminant, either from the lab or the isolated environment where the lab is located. It's somewhat like driving on an unfamliar road at night after just a few drinks. One doesn't intend for the multi-fatality crash to ensue, and indeed you could do this 20 times before "wooo--- what happened" spoils your day. Only in this case the experimenters are watching the car not in it.

I'm sure there are other non-conspiracy whacko events which might have occurred.

Personally I believe Nature to be far trickier than our inventions, but it's not impossible that human shennangans had a role in tickling nature to show its claws. Note that Royal Free Hospital disease appears to have no connection to any such experiments in the US.

In seriousness, beyond the problems of collection, storage and processing that are likely plaguing the negative studies, have PCR differences fully been ruled out? Have any of these 0/0 studies shown that their primers are capable of amplifying wild, clinical viral strains and not just synthetic strains?

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Hey guys.. the Boston-based study did not use the same methods as the Lombardi et al study.. not by a long shot. For starters, from that study:

In other words, they used the Reeves Empirical Definition to select their cohort of CFS patients. They also stated that of that cohort, more than 20% did not have "CFS" symptoms on a daily basis.

They also used VP62 synthetic clone both to calibrate their PCR assay and for positive controls. Apart from using the Lombardi primer sets for nested PCR, their study used a very different methodology from the WPI's... it's not even close. It was, however, very similar to the methodologies used in the other zero/zero studies, especially the CDC's.

asleep - PCR differences have not been ruled out (if the Blood Working Group has, they haven't made it public); all of the negative studies used synthetic clone to test their PCR assay, and did not show whether their assay could detect wild type virus.. although conveniently unpublished results by the Dutch XMRV team noted that their assay did turn up positives in samples from the WPI (I think those may have included controls, though). The CDC has been unable to detect virus in positive samples from the WPI or from Lo's group at the FDA.

That full text and those of the other recent publications are in the Research Archive at mecfsforums.com.. if you PM one of the admins, they'll probably get you a copy. I'll post the one you asked for in this library if I can.

My leanings have always been over the top in support of WPI. Now having said that is there a simple answer to this question: In the Science study for the healthy controls did they collect the sample in the same type of heparin coated test tube or not?

If they didn't, well then I have big doubts about xmrv. It would be great to find out that all test tubes used in the Science study even came from the same batch of tubes, not some from prior years or batches. Oh the humanity if we find out healthy controls go in a clear no preservative test tube and CFS blood goes in the heparin tube.

Somebody help me out and please quote where you know the info on how the controls were handled to the detail of what was in the test tube when it was empty.

In the Science study for the healthy controls did they collect the sample in the same type of heparin coated test tube or not? If they didn't, well then I have big doubts about xmrv. It would be great to find out that all test tubes used in the Science study even came from the same batch of tubes, not some from prior years or batches. Oh the humanity if we find out healthy controls go in a clear no preservative test tube and CFS blood goes in the heparin tube.

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It's very, very hard for me to credit that as a likely scenario. Did they all just not notice, or ignore, a systematic difference in the tubes used for blood collection? Did Lo/Alter make a similar mistake in blood collection? How would that explain the 4% in the healthy controls (unless of course one or two of them got the hep tubes)? Did all the PC positive studies also use disproportionately more hep tubes for the PC samples? Did they all use 3-10% hep tubes for the healthy controls? And with this emerging now as a clear area of controversy, have the WPI kept quiet after realising this? All those factors taken together rather beggar belief. But the strongest counter-argument I can think of right now is that the CDC still couldn't detect XMRV from the WPI or Lo/Alter positive samples: if those samples were hep-contaminated, the CDC should have found that.

I'm sure we could stack up more and more reasons, but I find it rather far-fetched. I can understand why you get jittery though, as this whole business continues to drag on and on...

I have to review LeGrice's talk as I was semi-out of it at the time but Houghton said during the Science summing up part that Lo/Alter found a PMRV and so did Maureen Hanson (when he was trying to say only WPI has found XMRV). This is the first I've heard of the Hanson study results -- the scientist in upstate NY who is working with David Bell -- did anyone remember or can confirm this too?

It's very, very hard for me to credit that as a likely scenario. Did they all just not notice, or ignore, a systematic difference in the tubes used for blood collection? Did Lo/Alter make a similar mistake in blood collection? How would that explain the 4% in the healthy controls (unless of course one or two of them got the hep tubes)? Did all the PC positive studies also use disproportionately more hep tubes for the PC samples? Did they all use 3-10% hep tubes for the healthy controls? And with this emerging now as a clear area of controversy, have the WPI kept quiet after realising this? All those factors taken together rather beggar belief. But the strongest counter-argument I can think of right now is that the CDC still couldn't detect XMRV from the WPI or Lo/Alter positive samples: if those samples were hep-contaminated, the CDC should have found that.

I'm sure we could stack up more and more reasons, but I find it rather far-fetched. I can understand why you get jittery though, as this whole business continues to drag on and on...

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Good points. It seems an exhaustive explanation by WPI into their exact methods would help my wavering. I just had my doc visit with dr. L app and 6 months prior I "donated" to his practice my wpi test tube kit since I decided not to drop $500 I didn't have. He opened the kit and studied the test tube. Now a week ago we are chattinga bout how things are not any clearer than 6 months prior and I mentioned this exact thing, WPI wouldn't have used different tubes for their different groups, sick and nonsick, and we both started at each other for 30 seconds and shook our heads and said nahh that just can't be. I gotta say during that 30 seconds though it was mighty uncomfortable contemplation going on.

I'd have to think that if they did use different tubes that would be more like deception than an innocent mistake. They couldn't fail to notice a procedural difference like that, using different tubes for the control samples and special infected ones for the patients - especially not a mistake that coincidentally happened to make the experiments all work out, and was repeated by others, including the prostate cancer positive studies: all those people can't possibly be using different tubes for the different groups and just not notice.

And any kind of deception theory wouldn't make any sense to me either because they would have to know they would be found out eventually given the intense interest. Apart from which it would be unbelievably callous to the patient community to do all that as a deliberate deception and they really, really don't come across that way. And note also that they have found XMRV in older samples too, unless I'm mistaken, and Lo/Alter definitely have found the PMRVs in samples 15 years apart. So none of this adds up that way to me.

The only explanation that makes any sense to me is that in order to detect XMRV you have to use the right methodology, and there are unexpected methodological requirements for successful detection, including the appropriate means of collection and storage of the blood. Since none of the negative studies have replicated the WPI's methodology, I don't see any reason to look any further than that for an explanation until they do so.

Despite repeated public comments about the importance of using the right tubes, unless I'm mistaken none of the negative studies have used the tubes recommended, or at least I don't think any of them have stated that they have done so. I don't think anybody has covered that sort of detailed information about blood collection and storage in their papers - I may be wrong, but I haven't seen any such detail in the negative papers. The WPI seem to have provided more detail about that sort of stuff than everybody else has. Sure one feels like one wants to have those detailed assurances, but I feel much more strongly that I need assurances demonstrating that the negative studies are actually capable of detecting XMRV at all.

In other words, they used the Reeves Empirical Definition to select their cohort of CFS patients. They also stated that of that cohort, more than 20% did not have "CFS" symptoms on a daily basis.

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What the... I would love to have CFS where I don't have daily symptoms. Where can I sign up?

Anyway, I will echo what I said in another thread. I just can't believe all these scientists don't want to find XMRV or are incompetent. There must be something else going on here. Still have high hopes about XMRV, but the negative studies just keep on piling...

Mark to play the advocate (I really believe the same way as you) I would see a scenario more like WPI really believes they've got XMRV present in CFS patients and discovering maybe some of the antagonists have a few points about heparin lined tubes, maybe "some" of their results might be affected in their view but they'll adjust for the heparin complaint and in their view still find XMRV in CFS. Nothing like a planned sinister motive, work and learn, adjust and keep believing in their theory.

I really wish WPI would state clearly how they did their lab procedures in order to settle in a dummy's mind like mine this once and for all. Failing to do this just worries me more the more time passes.

As for finding xmrv in old stored blood somebody on the board mentioned the other day that Defritus used heparin lined storage containers. So there you go again to your point of old blood containing xmrv, one could argue the old heparin batch had xmrv back then too.

Yes, i'm really, really waiting for that. And Dr. Lipkin's work. Those negative studies are making me nervous. But i find it hard to believe a 0/0 result. And also that all of the positive ones have been wrong. Are there any news about the Blood Working Group? I think they should be quite far advanced in their program by now.

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I think there are four key studies - the Blood Working group, Singh, the CAA/Glaxo Smith Kline study and Lipkin.

Dr. LeGrice told me he thinks the mouse DNA (contamination) issue will be resolved within 3 weeks! The Blood Working Group will announce findings as soon as they get - they know the patients are suffering under all this controversy. Their assay will hopefully be done by XMAS or early next year.

I'd have to think that if they did use different tubes that would be more like deception than an innocent mistake. They couldn't fail to notice a procedural difference like that, using different tubes for the control samples and special infected ones for the patients - especially not a mistake that coincidentally happened to make the experiments all work out, and was repeated by others, including the prostate cancer positive studies: all those people can't possibly be using different tubes for the different groups and just not notice.

And any kind of deception theory wouldn't make any sense to me either because they would have to know they would be found out eventually given the intense interest. Apart from which it would be unbelievably callous to the patient community to do all that as a deliberate deception and they really, really don't come across that way. And note also that they have found XMRV in older samples too, unless I'm mistaken, and Lo/Alter definitely have found the PMRVs in samples 15 years apart. So none of this adds up that way to me.

The only explanation that makes any sense to me is that in order to detect XMRV you have to use the right methodology, and there are unexpected methodological requirements for successful detection, including the appropriate means of collection and storage of the blood. Since none of the negative studies have replicated the WPI's methodology, I don't see any reason to look any further than that for an explanation until they do so.

Despite repeated public comments about the importance of using the right tubes, unless I'm mistaken none of the negative studies have used the tubes recommended, or at least I don't think any of them have stated that they have done so. I don't think anybody has covered that sort of detailed information about blood collection and storage in their papers - I may be wrong, but I haven't seen any such detail in the negative papers. The WPI seem to have provided more detail about that sort of stuff than everybody else has. Sure one feels like one wants to have those detailed assurances, but I feel much more strongly that I need assurances demonstrating that the negative studies are actually capable of detecting XMRV at all.

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Dr. Le Grices statement that the NCI is asking researchers to give extremely detailed reports to the NCI - which they will post on their website - indicates that they are looking at aspects of the methodological process that are not usually reported in scientific papers. I think Mark is completely correct - there are 'unexpected methodological requirements for successful detection'. I think it was clear a while ago that whatever was causing the disparate results was 'unusual'.

I don't see any reason any retrovirologists would not want to find XMRV/ I think we should plant the idea that any retrovirologist would want to find XMRV DEEPLY in our brains. This is like an astronomer finding a new planet of something like that - something they can study for the next couple of decades. They all want to find this retrovirus.. They want to find it in AIDS, in people with organ transplants - they don't give a hoot about CFS, CFS is entirely secondary - CFS is simply the vehicle they could use to find what they've always wanted to find - another infectious retrovirus. The key for them is the virus - not the population they are finding it in.

The problem, I think, is that XMRV is not showing up using standard methods.The inability of retrovirologists to use their standard instruments to find the pathogen is what has lead to the questions about contamination - and every other step of the process. That is why they are looking at blood storage, test tubes, etc. - things they don't usually worry about because they don't need to.

Their willingness to go unusual lengths to look at every aspect of the process is the promising part for me and that is what didn't happen with the De Freitas. The reason it's happening now, I think, is that the results are not easy to explain by contamination either. You have to have a string of kind of bizarre circumstances to explain away the entire Science paper: a difficult to find contaminant AND antibodies cross-reacting to a DIFFERENT virus (which is similar). What are the chances of those two things happening? Plus you just happen to find the antibodies or positive PCR tests in CFS patients and not in controls.......That's hard to explain!

They have a very solid first study they have to account for - I think that is why they are going to such lengths.

I just can't believe all these scientists don't want to find XMRV or are incompetent.

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Jemal, I agree that this is certainly not true of all the scientists involved in these negative studies. However, one or both of these accusations are certainly true of many of these scientists. For example, both Reeves and Wessely preemptively stated (under the thinnest veil of objectivity conceivable) that they would not find the virus. So it's not unreasonable to question the underlying motivations of studies directly or indirectly involving them.

Then there's the sordid history of the CDC and psychologists (the new Cooperative Diagnostics study was overseen by a psychosomatisizer). Can a we really believe that a study involving them represents a genuine attempt to find XMRV? And as these 0/0 studies pile up, so does the plausible deniability and wiggle room for bad motives: "See, no one else could find it, so my study [where I waved a stick I found in my front yard over test tubes to see if they turned green] must also be valid! To the contamination mobile, Dr. Reeves!"

And finally, many of the remaining negative studies have explicitly drawn woefully illogical conclusions (as has been pointed out by others ad nauseum). To me this definitely sets off alarm bells regarding motivations and/or competence. If you can't form a logical conclusion, can the rest of your study be that well designed? Why would competent, well-intentioned scientists overreach so drastically in a way that is indistinguishable from an attempt to bury this finding?

Happy to hear maybe something to read in 3 weeks, give or take a month. Cort nice to see you on the webcast, that was pretty cool. This sure is a complicated puzzle with many divergent interests involved. Patent stuff, scientists first to get credit, reputations and accolades, criticism, who gets what in that category. Maybe WPI is just tired of the contamination issue and is passed it all knowing that they are right. Maybe they found a smidgeon of contamination and know it's nothing to worry about but admitting it would kill the ongoing efforts. Maybe they have a big oops and are scrambling, like when critics point out that Lo/Alter didn't find xmrv.

I don't get comfort on the dark side thoughts of contamination when it's pointed out that everybody wants to find xmrv, fits with WPI wanting to find it (/they did find it). Okay so I have a fogged too far gone comprehension of science so I boil it down to finding some very simple comforts and one comfort I could really latch on to is a statement by WPI on how they segregated healthy from sick in their lab. I want to hear it down to the tubes they used on each.

Sometimes trying to read a face gets you somewhere, sometimes not and I do like to scramble (google crawler paranoia) my well known cfs doc's name as to not attribute anything not meant to be conveyed to me but while talking over heparin with him I saw a flash of worry for maybe 5 seconds during contemplation, then back to naah it just couldn't be. Wish I could get that out of my head.

There is no reason in the world that a retrovirologist will not want to find XMRV! I think we should plant that idea DEEPLY in our brains.

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Really? No reason? I have to say that I think this is very naive, esp. considering even a cursory understanding of the history of CFS. The idea that one's intentions/motivations must never be questioned and could never be rotten is too faith-based cultish for me. History is rife with examples where bad intentions have prevailed for non-trivial periods of time.

In its most basic form, these negative studies set out to do something (detect XMRV) that someone else (WPI) claims to have done, but they came up empty. There are only two general reasons for the discrepancy: 1) the original claim is false (e.g. contamination, false postives, etc) or 2) the followup attempts are inadequate in some fashion (e.g. issue w/ method or process). I believe that if one is both competent and well-intentioned, then he/she would willingly and proactively acknowledge and investigate both possibilities. A competent and well-intentioned person doesn't (as has happened in most of these cases) state unequivocally that the first option is the only possibility and then launch into a PR crusade to discredit the original claim. Perhaps this is explained by an utter lack of humility on their part, a failure to even consider that their efforts are inadequate. However, I would argue that letting your ego interfere with good science is fundamentally a form of incompetence.